Purpose:
The expression of human leukocyte antigen (HLA) on tumor cells may influence the immunological recognition and response. Malignant cells often change their HLA class I antigens to escape immunosurveillance. To examine whether immunotherapy is possible in conjunctival melanoma and retinoblastoma we investigated the HLA class I surface expression by using allele-specific antibodies in flow cytometry. We furthermore analyzed the effect of interferon gamma (IFN-γ) stimulation on HLA expression.

Methods:
Three conjunctival melanoma (CRMM1, CRMM2 and CM2005.1) and three retinoblastoma (Rb116, Rb125 and Rb143) cell lines were HLA gene-typed at the Department for Immunohematology and Blood Transfusion (IHB), The Netherlands. Cells were grown in culture in the presence or absence of recombinant IFN-γ (100 and 200 U/ml) for 48 hours and prepared for flow cytometry. Based on the HLA-typing, HLA allele-specific monoclonal antibodies against class I were selected. Flow cytometry was performed and cellular surface HLA-expression measured. For Western blot analysis, a lysate was made to determine the expression of MHC class I.

Results:
We analyzed the HLA allele-specific expression on three conjunctival melanoma and three retinoblastoma cell lines by flow cytometry. In general, a lower expression of antigen specific and allele-specific HLA class I expression was observed in both types of malignancies. In one retinoblastoma cell line (Rb143) no class I expression was observed without IFN-γ stimulation but it was restored after incubation with IFN-γ. Two of the three conjunctival melanoma (CRMM1 and CRMM2) cell lines showed loss of expression of specific HLA class I alleles (respectively HLA-A2 and HLA-B44) which did not recover after IFN-γ stimulation.

Conclusions:
We were able to determine HLA expression on three new retinoblastoma cell lines and three conjunctival cell lines, and found a defect in expression of particular HLA specific alleles which were not restored by IFN-γ stimulation. This loss of antigen expression may help ocular tumors to escape the immune response and complicate the development of immunotherapy.